Many species of bacteria accumulate granules of polyesters composed of hydroxyacyl monomers which serve as carbon reserves. The occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates has recently been reviewed (Anderson, A. and Dawes, E. A., Microbiol. Rev. 54:450-472 (1990)). The most commonly found compound of this class is poly(D(-)-3-hydroxybutyrate). However, some species accumulate copolymers of different hydroxyalkanoates such as 3-hydroxypentaneoate (Wallen, L. L. and Rohwedder, W. K., Environ. Sci. Technol. 8:576-579 (1974)). At least 11 short-chain 3-hydroxyacids are found as components of polymers from marine sediments. Studies of polyhydroxyalkanoate production in Alcaligenes eutrophus have shown that when the bacteria are cultivated in a medium with only glucose as a carbon source, only PHB is accumulated. However, when both glucose and propionic acid are provided as carbon sources, the bacteria accumulates random copolymers of 3-hydroxypentanoate and 3-hydroxybutyrate (Holmes, P. A., Phys. Technol. 16:32-36 (1985); Holmes, P. A., Wright, L. F. and Collins, S. H. European Patents 0 069 497, January 1983 and 0 052 459, December 1985). In addition, when A. eutrophus is supplied with various other carbon sources, polyesters containing 4-hydroxybutyrate and 5-hydroxyvalerate monomers are produced (Table I in Anderson, A. J. and Dawes, A. E., Microbiol. Rev. 54:450-472 (1990)). Thus, it appears that the composition of the polymer is regulated to some extent by the availability of alternative substrates for the enzymes which catalyzed synthesis of the polymer from monomers.
PHB accumulates in bacterial cells as granules of approximately 0.24 to 0.5 .mu.m in diameter. On the basis of measurements of the molecular weight of PHB monomers, each granule has been estimated to contain a minimum of 1,000 polymer chains. The granules have been proposed to possess a membrane coat composed of lipid and protein representing approximately 0.5 and 2%, respectively, of the granule weight (Anderson, A. and Dawes, E. A., Microbiol. Rev. 54:450-472 (1990)). The activity of the PHB synthase enzyme is thought to be associated with this membrane. The state of the PHB within the granule is a matter of substantial uncertainty. Recent evidence suggests that the polymer within the granules is in an amorphous state. It is not known what regulates the size of PHB granules in any organism.
In most organisms, PHB is synthesized from acetyl coenzyme A (acetyl-CoA) by a sequence of three reactions catalyzed by 3-ketothiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9), acetoacetyl-CoA reductase (hydroxybutyryl-CoA dehydrogenase; EC 1.1.1.36) and poly(3-hydroxybutyrate)synthase. The pathway is shown in FIG. 1. In Rhodospirillum rubrum, PHB is synthesized by conversion of L(+)-3-hydroxybutyryl-CoA to crotonyl-CoA to D(-)-3-hydroxybutyryl-CoA. The 3-ketothiolase has been purified from various PHB-synthesizing bacteria and has been studied in several species of higher plants. The role of the enzyme in higher plants is thought to be in the production of acetoacetyl-CoA for mevalonate production as well as in the degradation of fatty acids. The acetoacetyl-CoA reductase has been detected in a number of PHB-synthesizing bacteria. Several species, including A. eutrophus, appear to have two isoenzymes which differ with respect to substrate specificities and cofactor requirements. The NADH reductase of A. eutrophus is active with C4 to C10 D(-)- and L(+)-3-hydroxyacyl-CoAs, whereas the NADPH reductase is active with only C4 and C5 D(-)-3-hydroxyacyl-CoAs. An enzyme of this kind has never been reported in higher plants. PHB synthase activity has been detected in PHB-accumulating bacteria as both a soluble enzyme and as a granule-bound activity, depending on the growth conditions. Both forms of the enzyme have been partially purified but have not as yet been purified to homogeneity because of instability. The PHB synthases of A. eutrophus is specific for D(-)-enantiomers and when tested with 3-hydroxyacyl-CoAs, was shown to be active only with C4 and C5 substrates, consistent with the observation that only C4 and C5 3-hydroxyacid monomer units are incorporated into the polymer by this organism. The mechanism of PHB synthase action remains obscure. It is presumed that the chain transfer role played by the synthase must in some way control the molecular weight of the polymer produced, which is characteristic of a given organism. PHB synthase activity has never been reported in any plant.
Several groups of researchers have independently cloned, and expressed in E. coli, the genes involved in the biosynthesis of PHB by A. eutrophus (Slater, S.C., et al., J. Bacteriol. 170:4431-4436 (1988); Schubert, P., et al., J. Bacteriol. 170:5837-5847 (1988)). Recombinant strains of E. coli carrying a 5.2 kbp fragment from A. eutrophus were capable of accumulating substantial quantities of PHB as intracellular granules. The nucleotide sequence of the 5.2 kbp fragment was also independently determined by two groups (Janes, B. B., et al., In Dawes, E. A. (ed) Novel Biodegradable Microbial Polymers, Kluwer Academic Publishers, pp 175-190 (1990); Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264:15293-15297 (1989); Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264:15298-15303 (1989)). Analysis of the deduced amino acid sequences of the open reading frames, in conjunction with evidence based on genetic complementation studies, revealed that the 5.2 kbp fragment contained three closely linked genes encoding the three enzymes required for PHB production. A patent concerning the use of the cloned genes to overproduce the biosynthetic enzymes in bacteria has been filed (Peoples, O. P. and Sinskey, A. J., Int Patent WO 89/00202, January 1989).
Certain species of bacteria have the ability to excrete enzymes and degrade PHB and related polyhydroxyalkanoates (Reviewed in Anderson, A. and Dawes, E. A., Microbiol. Rev. 54:450-472 (1990)). Because of the prevalence of these bacterial species in many natural environments, PHB is rapidly degraded in soil and activated sludge. Thus, PHB and related polyhydroxyalkanoates are of interest as renewable sources of biodegradable thermoplastic. Industrial PHB production from large-scale cultivation of bacteria began in 1982. The PHB produced in this way is marketed by ICI plc under the trade name Biopol. However, because of the costs associated with growing and harvesting large cultures of bacteria, the PHB is much more costly to produce than polymeric materials such as starch which are accumulated to high levels in many species of higher plants. Therefore, it may be advantageous to develop, by genetic engineering, lines of higher plants which accumulate PHB.